The process of sporulation in the Gram-positive bacterium Bacillus subtilis will be used as a model system for investigating the regulation of growth and developmental gene expression in procaryotes. The proposed work focuses primarily on two critical areas: signal transduction mechanisms responsible for sensing and responding to nutritional stress and mechanisms responsible for establishing compartment-specific programs of gene expression in the developing sporangium. The long-range objective is to understand fundamental aspects of physiology and gene regulation that will have important implications for the use, manipulation and control of a wide range of procaryotic microorganisms, including species of public health and industrial relevance. Much of the proposed work concerns two regulatory proteins, SpoOA and SpollE. SpoOA is a phosphorylation-activated transcription factor which belongs to the "response regulator" superfamily of bacterial signal transduction proteins. A key objective of the work is to identify and recover genes whose products sense or transmit nutritional information through the signal-transduction pathway that controls the phosphorylation state of SpoOA. This will involve, in part, the development of a new class of transposon delivery vector which is expected to facilitate saturation-mutagenesis in B. subtilis and related bacteria. Another objective is to define and understand the mechanistic role of SpoOA domains responsible for DNA-binding and transcriptional activation. This effort will be aided by the recent characterization of SpoOA homologs present in diverse Bacillus and Clostridium species, which has revealed regions of the protein that play critical roles in structure or function. This and other information will be used to target specific regions of SpoOA for mutagenesis, creating primary mutations which will be employed in the selection for intragenic and extragenic suppressors. Mutant proteins will also be subjected to extensive biochemical analysis, which is expected to reveal whether the protein functions as a monomer, dimer or higher-order multimer and to determine the specific functional nature of the phosphorylation-activated step. SpollE is a protein required for normal partitioning of the developing sporangium into separate compartments with separate programs of gene activation and regulation. Available evidence suggests that a SpollE-determined feature of the sporulation septum may be responsible for generating a signal or creating a condition in the forespore compartment that activates the function of a compartment-specific RNA polymerase sigma factor. We propose to determine the intracellular location, membrane-spanning topology and function of the SpollE protein.